SUMMARY Ras signaling promotes proliferation, cell survival, and differentiation and is implicated in cancer. To devise effective therapeutics, it is vital to understand how Ras is regulated and how to block its activity. My lab discovered that modifying Ras by attaching the small protein ubiquitin restricts both wild-type and oncogenic forms of Ras. Impairing Ras ubiquitination in vivo in Drosophila led to striking effects on cell proliferation, cell survival, developmental patterning, and organismal longevity, reflecting a role for Ras ubiquitination in development, tumor suppression, and survival. We identified the Ras E3 (the ubiquitin ligase that adds ubiquitin to Ras) as Rabex-5 (also called RabGEF1). Intriguingly, we discovered that Rabex-5 also regulates the Notch oncogene. The power of Drosophila genetics and the well-established paradigm of studying Ras in Drosophila make this system ideally-suited to characterize these phenomena in a multi-cellular context. The goal of this proposal is to use in vivo Drosophila studies and in vitro biochemical analysis to elucidate the biological significance and molecular mechanisms of Ubiquitin Pathway regulation of Ras and Notch signaling. Defining the molecular mechanism(s) of Ras ubiquitination will yield key insights into fundamental biology and will reveal novel points of therapeutic entry in cancer. We made initial observations regarding spatial and temporal regulation of Ras ubiquitination. Consistent with data that Rabex-5 is lost or decreased in various cancers, our loss-of-function studies unambigiously indicate a tumor suppressor role for Rabex-5 by inhibiting Ras and Notch signaling. We hypothesize that Rabex-5 restricts Ras and Notch and acts in tumor suppression via its E3 domain. By complementing in vivo Drosophila work with in vitro biochemistry, we are uniquely positioned to make significant advances into conserved, fundamental mechanisms of regulation and coordination of Ras and Notch oncogenic signaling.